Crystallizing the Architects of Diversity

Phong Lee | phong.lee@yale.edu December 27, 2008

Structure of the Group II splicing intron

“This project culminates many years of study. It confirms nearly two decades of scientific research,” remarked Anna M. Pyle, William Edward Gilbert Professor of Molecular Biophysics and Biochemistry.

Pyle recently led a group of researchers who solved the structure of the group II splicing intron, a molecule responsible for diversifying the world’s most ancient organisms.

The crystal structure unlocks a wealth of information revealing previously unfathomable RNA interactions and provides possibilities for critical advances in the field of gene therapy.

Group II introns are mobile genetic elements that shaped the genomes of various organisms including bacteria, fungi, and plants. These molecules have the ability to catalyze a diverse group of reactions with DNA and RNA.

They excise themselves from RNA molecules, ligate the RNA pieces back together, and then re-insert themselves into new target sequences. “Group II introns create new gene structures, thereby allowing a single gene to encode multiple different proteins,” says Pyle. “These molecules are the accidental architects of diversity.”

Pyle became fascinated with group II introns and particularly their structure when her academic career began over sixteen years ago.

“The reason [a Group II intron] is interesting is because it is descended from an ancient catalyst of diversity that influenced the evolution of all life forms. This molecule and other mobile elements represent engines for change, causing genomes to be in continual flux.”

She has relied on a variety of sophisticated biomedical techniques to study and model group II introns. Pyle noted that one of the most elegant techniques she employed was a powerful form of chemical genetics in which she mutated RNA on the atomic level and deduced which atoms caused certain functions.

This form of chemogenetic suppression was invented by Scott Strobel, a Henry Ford II Professor of Molecular Biophysics & Biochemistry and one of Pyle’s colleagues. She coupled this approach with computational strategies, and in 2005, she indirectly solved the structure of a group II splicing intron.

However, she needed a definitive image of the intron and turned to x-ray crystallography. Finding a group II intron to crystallize was extremely difficult because the molecule seldom splices in vitro.

The only established method for in vitro splicing of the group II intron required warm temperatures and high concentrations of salt, conditions which were unfit for the production of crystals for structural studies.

She searched for variants of the group II splicing intron and found a suitable match in the deep sea bacterium, Oceanobacillus iheyensis, which thrives in extremely alkaline environments. The intron self-spliced in the laboratory and formed stable structures that could be readily crystallized for analysis.

Pyle was then able to employ x-ray crystallography, which revealed the structure of the elusive group II intron. After having deduced the structure of the group II intron, Pyle hopes to use the information to make additional advances in molecular biology and the understanding of evolution.

Group II introns can exist in multiple states and Pyle hopes to capture the intron in those various states and deduce its structures. “If you pick a good problem there are a lot of things you can learn along the way.” Indeed, her quest to study and model group II introns continues.